Journal of Controlled Release 138 (2009) 71–77

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Journal of Controlled Release

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GENEDELIVERY

Parameters affecting organization and transfection efficiency of amphiphiliccopolymers/DNA carriers

Caroline Roques a,b,c,d, Kawthar Bouchemal a,b, Gilles Ponchel a,b, Yves Fromes c,d, Elias Fattal a,b,⁎a Univ Paris Sud 11, UMR CNRS 8612, Franceb CNRS, Châtenay-Malabry, F-92290, Francec Inserm, U582, Institut de Myologie, Paris, F-75013, Franced UPMC Univ Paris 06, UMR_S582, IFR14, Paris, F-75013, France

⁎ Corresponding author. UMRCNRS8612, Centre d'EtudParis-Sud 11, 5, rue Jean-Baptiste Clément, 92290 Châtenay83 55 68; fax: +33 1 46 61 93 34.

E-mail address: elias.fattal@u-psud.fr (E. Fattal).

0168-3659/$ – see front matter © 2009 Elsevier B.V. Adoi:10.1016/j.jconrel.2009.04.030

a b s t r a c t

a r t i c l e i n f o

Article history:Received 2 March 2009Accepted 27 April 2009Available online 3 May 2009

Keywords:Gene transferAmphiphilic copolymersMicellizationStriated musclesDystrophic muscle

Amphiphilic block copolymers are attracting increasing interest in the field of gene therapy, especially fortransfection of striated muscles. However, little is known about the parameters affecting their transfectionefficiency in vivo. These copolymers can self-assemble as micelles in certain conditions. Since micellizationstrongly depends on the temperature and ionic content of the preparation medium, the present paper aimedat investigating the influence of these parameters in the context of gene delivery. We first assessed themicellization of pluronic L64 and tetronic 304 at various temperatures in water, saline or Tyrode's saltssolution. Pluronic L64 can form micelles at temperatures above 37 °C in water or at 37 °C in the Tyrode's saltssolution, in the range of concentration investigated. For tetronic 304, CMC was found to be far below theconcentrations used to transfer DNA. Pluronic L64 interacted with DNA only in the presence of micelles.Moreover, in vivo evaluation demonstrated that significantly improved transfection efficiency was obtainedat 37 °C in Tyrode's salts solution for pluronic L64 based formulations, compared to 4 °C and 20 °C. Suchdifferences were not recorded with tetronic 304. Finally, optimized formulations of both tetronic 304 andpluronic L64 were able to mediate efficient transfection in dystrophic muscles.

© 2009 Elsevier B.V. All rights reserved.

1. Introduction

Muscle tissue is an attractive target for the development ofinnovative therapeutics based on gene transfer. Both cardiac andskeletal muscles can efficiently express therapeutic proteins fromexogenous plasmids [1,2]. Furthermore, various skeletal muscles caneasily be accessed through direct intramuscular injections. However,large amounts of naked DNA must be administered in order to obtainrelatively low levels of protein expression.

Design anddevelopmentof genecarriersmediating safe andefficientgene transfer is thus required to attain therapeutic levels of proteinexpression. In such context, synthetic vectors emerge as an attractivestrategy given their versatility and their limited side effects compared toviral vectors. However, muscular tissue remains difficult to transducewith cationic lipids and polymers. In the past years, growing interest has

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focused on amphiphilic copolymers composed of poly(ethyleneoxide)(EO) and poly(propyleneoxide) (PO) blocks which have demonstratedtheir ability to safely and efficiently transfer nucleic acids to striatedmuscles [3–6]. While their in vivo efficiency has been established, theirmechanisms of action and the parameters influencing their efficacy arecurrently poorly understood.

Due to their amphiphilic properties, pluronics and tetronics exhibitthe ability to spontaneously self-assemble as micelles above a certainconcentration called critical micelle concentration (CMC). CMC can bereached at various concentrations depending in particular on thetemperature, the ionic content or, especially for charged compounds,the pH of the media used [7–15]. Although these properties areimportant characteristics of such polymers, the effect of medium andtemperature on their CMC, and subsequently their transfectionefficiency, has never been investigated.

Thus, our work has focused on the definition of the formulationparameters, which might affect the supramolecular structure ofamphiphilic copolymers displaying few or no charges. We alsoconsidered the influence of these modifications on the interactionsbetween the copolymers and plasmid DNA. Moreover, we assessedhow these parameters can impact on the in vivo efficiency of suchcarriers once transferring plasmid DNA to the skeletal muscles.

Fig. 1. Determination of the CMC of tetronic 304 in water at various temperatures byusing ITC. A sigmoid curve is obtained from the integration of the calorimetric peaksresulting from the titration of tetronic 304 inwater, whatever the temperature. A typicalcurve recorded at 37 °C is presented on panel A. The enthalpy of micellization(ΔHmicellization) can be calculated from the difference between the extrapolated linesrepresenting the start and the end of micellization (panel A). The CMC corresponds tothe maximum of the first derivative of the titration curve as shown on panel B. CMC andvarious thermodynamic parameters were determined from the titration curves and aresummarized on panel C. ΔGmicellization: free energy of micellization; ΔSmicellization:entropy of micellization.

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2. Materials and methods

2.1. Materials

A plasmid coding for the bacterial β-galactosidase (pCMV-βGal,7.2 kb, Clontech, France) was used throughout the study. The plasmidwas amplified in JM109 E. coli and purified using the Endofree PlasmidGiga Kit (Qiagen S.A, France) according to themanufacturer's protocol.Pluronic L64 and tetronic 304 were obtained from BASF and used asreceived.

2.2. Methods

2.2.1. Preparation of copolymer/DNA formulationsStock solutions of pluronic L64 and tetronic 304 were prepared in

water at concentrations of 2% (w/v) and 10% (w/v) respectively. Stocksolutions were stored at least 24 h at 4 °C before use. All formulationswere prepared by gently mixing equal volumes of suitably dilutedsolutions of polymer in water and DNA in Tyrode's salt solution (2X)and were allowed to equilibrate at least 30 min prior to use. Forexperiments evaluating the effect of temperature, all the componentsof the formulations were maintained at 4, 20 or 37 °C at least 30 minbefore their association and thereafter kept at constant temperatureuntil use. Final concentrations of tetronic 304 and pluronic L64 wererespectively 5% (w/v; corresponding to 30.3 mM) and 0.05% (w/v;corresponding to 0.17 mM).

2.2.2. Isothermal titration microcalorimetryAn isothermal calorimeter (MicroCal Inc., USA) was used to

determine both the critical micelle concentration (CMC) and theenthalpy of micellization of pluronic L64 and tetronic 304 [16].Typically, a syringe containing 283 µl of copolymer solution isprogressively diluted in the sample cell (cell volume: 1.441 ml)containing water, saline or Tyrode's salt solution and insulated by anadiabatic shield. For each experiment, 2.5 to 10 µl of copolymersolution were delivered over 20 s with a 600 s interval betweeninjections to allow complete equilibration. The sample cell wassubmitted to continuous mixing at 220 rpm throughout the experi-ment [16]. Each titration was performed three times to ensurereproducibility of the results.

To assess interactions between the amphiphilic copolymers andthe DNA, a solution of pluronic L64 was titrated into a plasmid DNAsolution contained in the sample cell. Both pluronic L64 and DNAwerediluted in Tyrode's salt solution. 10 µl of pluronic L64 was deliveredover 20 s with a 400 s interval between injections to allow completeequilibration. The heat of dilution of pluronic L64 was obtained bytitrating the copolymer diluted in Tyrode into a solution of Tyrode. Theheat of interaction between pluronic L64 and DNA was determinedafter subtracting the heat of dilution of pluronic L64.

Evaluation of tetronic 304/DNA interactions was carried out bytitrating a copolymer solution in water into a DNA solution in Tyrode2X. The parameters were the same as for those used to study pluronicL64/DNA interactions. The heat of dilution of tetronic 304 wasobtained by titrating the copolymer in water into a solution of Tyrode2X. The heat of interaction between tetronic and DNAwas determinedafter subtracting the heat of dilution of tetronic 304.

2.2.3. In vivo transfection efficiencyIn vivo experiments were carried out onwild type Syrian hamsters

(strain Rj:AURA; Elevage Janvier, France) as well as CHF147 Syrianhamsters [17]. CHF147 hamsters were bred at the animal facility of theInstitut de Myologie. All animal studies were approved by the Institutde Myologie review board. To evaluate transfection efficiency of thevectors, direct intramuscular injections were performed on bothtibialis anterior and quadriceps muscles. Prior to administrations,animals were anesthetized via intraperitoneal administration of

ketamine (75 mg/kg), xylazine (15 mg/kg) and midazolam(0.75 mg/kg). 150 µl of each formulation containing 200 µg of DNAwere administered in each muscle (n=at least 3 for each muscle andeach condition). One week after administration, animals weresacrificed with an overdose of anesthetics. Muscles were harvestedand immediately plunged into an isopentane bath cooled with liquidnitrogen. On eachmuscle, 8 µm thick cryo-sections were performed atten different levels separated from 800 µm. X-Gal revelation andHaematein/Eosin counter-staining were carried out on the cryo-sections after 5 min fixation in paraformaldehyde (4% w/v)/glutaraldehyde (0.2% w/v) solution. Evaluation of the transfectionefficiency depending on the medium and the temperature wereperformed on wild type animals. CHF147 hamsters received pluronicL64 and tetronic 304 gene carriers prepared in Tyrode's salts solutionand thermostated at 37 °C.

3. Results

In this study, we investigated tetronics and pluronics copolymersas candidates to achieve efficient and safe gene delivery to the striatedmuscles. They both consist in amphiphilic block copolymers and thusdisplay the ability to formmicelles under certain conditions. Althoughtheir micellization has been extensively studied, there is a lack ofinformation about their structural organization in the media used toperform gene transfer within the polymer range of concentrationemployed to this purpose.

Micellization of the tetronic 304 was assessed in water byisothermal titration microcalorimetry (ITC) (Fig. 1).

After analysis of the data obtained from the titration curves (asrepresented on Fig. 1A and 1B), the CMC, ΔHmicellization, ΔGmicellization

and TΔSmicellization were calculated for each temperature investigated

Fig. 2. Influence of the temperature on the interactions between unimers of pluronicL64 as assessed by ITC (panel A). Titration of the copolymer in water was performed atthe following temperatures: 4 °C (×), 20 °C (●), 25 °C (○), 37 °C (x), 39.5 °C (1), 42 °C(□), 44.5 °C (▲), 46 °C(✳) and 50 °C (■). With pluronic L64, no sigmoid curves could beobtained, but the enthalpograms are clearly separated into two parts (panel B;enthalpogram recorded at 44.5 °C). The range of concentration containing the CMC thuscan be determined from the lines presented on panel B. The CMC is assumed to bereached between the start of the transition (ST) and the end of transition (ET). The CMCreported on the graph is corresponding to data from the literature [7].

Fig. 3. Influence of medium and temperature on the micellization of pluronic L64 asdetermined by ITC measurements. Titration of pluronic L64 was first performed at 37 °Cin water (□), saline (✳) or Tyrode's salts solution (■) (panel A). Interactions betweenthe unimers were only recorded with the Tyrode's salts solution at the settledtemperature in the range of pluronic L64 concentrations tested. Micellization ofpluronic L64 was then carried out in Tyrode's salts solution at 4 °C (▲) and 20 °C (●)and compared to the titration curve obtained at 37 °C (■). Titration curves recorded at4 °C and 20 °C are identical and no interaction occurred between the unimers at thosetemperatures in Tyrode's salts solution, contrarily to 37 °C.

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(Fig. 1C). Micellization of tetronic 304 was demonstrated to be atemperature dependent process. Increasing the temperature from 4 °Cto 37 °C led to a decrease in the CMC of tetronic 304 and an increase inthe enthalpy of micellization. This indicates that the aggregationprocess was favoured when increasing the temperature. The freeenergy of micellization (ΔGmicellization) was found to be negative,whatever the temperature, thus indicating that thermodynamicallystable micelles are spontaneously formed. The calculated entropy ofmicellization (ΔSmicellization) was positive and increased with increas-ing temperature from 4 °C to 37 °C. This gain in entropy correspondedto the facilitated release of water molecules from the hydrophobic POresidues of the unimers (e.g. individualized copolymer molecules)during the micellization process. With tetronic 304, the CMC could bereached in water at low concentrations of copolymer, even at lowtemperatures. At the concentration used to perform administration ofnucleic acids in the muscle tissue, e.g. 30 mM, tetronic 304 is thusforming micelles whatever the temperature in water. As the increasein ionic strength decreases the CMC of tetronic 304, the concentra-tions of the copolymer used in our experimental set-up are beyond theCMC of tetronic 304 in both saline and Tyrode's salts solution.

Organization of pluronic L64 into micelles was also investigated inwater at increasing temperatures by ITC (Fig. 2).

Contrarily to tetronic 304, titration curves obtained with pluronicL64 did not display a sigmoid shape. Thus, the CMC was more difficultto determine precisely for this copolymer. However, the analysis of theenthalpogram of pluronic L64 inwater led to distinguish two separateheat effects for temperatures above 37 °C (Fig. 2A). Initially, themicelles of pluronic L64 contained in the syringe broke up wheninjected in the sample cell and subsequently the copolymer existed asunimers. First part of the titration curve thus resulted (i) from the heatof dilution of the copolymer in water and (ii) from additional heateffect due to micelles breakup. The second part of the titration curves,e.g. above the CMC, resulted only from the dilution of the micelles,explaining the reduce value ofΔH. Between these two regions, there isa transition corresponding to the progressive aggregation of theunimers. The titration curves were analyzed as suggested by severalauthors [18,19]. Linear fits of the data allow to determine the start oftransition (ST, corresponding to the last experimental point on thefirst fit) and the end of transition (ET, corresponding to the firstexperimental point on the second fit). The CMC is assumed to bereached between ST and ET. As shown on Fig. 2B, the range ofconcentration comprising the CMC determined with this method is ingood agreement with the data from the literature giving the CMC ofpluronic L64 as assessed with the dye solubilization technique [7]. Wecould also observe that this range of concentration decreased as afunction of temperature (Fig. 2A), indicating that the CMC isdecreasing with temperature. More importantly, no interactionoccurred between the unimers at temperatures below 42 °C. Thisindicates that, in the range of concentration tested, no micellizationtook place in physiological conditions.

Table 1Enthalpy of interaction between amphiphilic copolymers and DNA depending on thetemperature.

Temperature (°C) ΔHInteraction tetronic304/DNAa (kJ/mol)

ΔHInteraction pluronicL64/DNAb (kJ/mol)

4 0.20 020 0.19 037 0.19 2.13

a For tetronic 304, CMC was reached independently of the temperature.b At 4 and 20 °C, concentration of pluronic L64 was below the CMC at these

temperatures. At 37 °C, concentration of pluronic copolymer was above the CMC.

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In a second step, we investigated the interaction between thepluronic L64 unimers at 37 °C in various media and in the Tyrode'ssalts solution at various temperatures (Fig. 3).

To allow a more precise determination of the CMC of pluronic L64in those conditions, smaller amounts of pluronic were injected intothe sample cell (2.5 µl instead of 10 µl). In water or saline, noformation of micelles was recorded at 37 °C, in the range ofconcentrations used (Fig. 3A). Conversely, when introducing pluronicL64 in the Tyrode's salts solution, unimers interactions could beobserved. However, interactions between the unimers of pluronic L64

Fig. 4. Effect of the medium on the transfection efficiency of formulations based onpluronic L64. PluronicL64/DNA formulations were prepared in water (A), saline (B), orTyrode's salts solution (C) and directly injected in quadriceps muscles of wild typeSyrian hamsters. For each muscle, 200 µg of DNAwere administered. Oneweek after theinjections, muscles were harvested and revelation of the β-galactosidase activity wasperformed on the cryo-sections.

were recorded in the Tyrode's salts solution at 37 °C, but not at 4 °C or20 °C in our experimental set-up (Fig. 3B).

As we demonstrated that the supramolecular organization ofamphiphilic copolymers is strongly influenced by the temperatureand the medium used, we wanted to further evaluate how theseconditions could influence the interactions between amphiphiliccopolymers and DNA. Enthalpy of interaction between pluronic L64 ortetronic 304 and DNA were thus investigated by ITC in Tyrode's saltssolution at various temperatures (Table 1).

Tetronic 304 is able to interact with the plasmids, and the enthalpyof interaction remains constant in the range of temperaturesconsidered. These results are consistent with the data onmicellizationof tetronic copolymer insofar as the concentration of tetronic 304 issuperior to the CMC, whatever the temperature.

On the contrary, no interactions could be recorded in Tyrode's saltssolution between pluronic L64 and DNA at 4 °C and 20 °C. Theseconditions did not lead to the micellization of the amphiphiliccopolymer, as presented earlier. When pluronic L64 self-assembledasmicelles, e.g. at 37 °C, interactions occurredwith the DNA. However,these interactions are not leading to the condensation of the DNA, asthe profiles of the DNA/pluronic L64 systems as recorded by dynamiclight scattering are not modified when the temperature is raised from

Fig. 5. Effect of the temperature on the transfection efficiency of pluronic L64 andtetronic 304 gene carriers. Formulations based on pluronic L64 and tetronic 304 wereprepared at 4 °C, 20 °C or 37 °C in Tyrode's salts solution and administered throughdirect injection in tibialis anterior and quadriceps muscles. 200 µg of DNAwere injectedin each muscle. Muscles were harvested 1 week after administration and ten differentlevels of cryo-section were achieved on each muscle. Representative sections of tibialisanterior muscles are presented for each polymer and each temperature on panel A. Foreach level where cryo-sections were performed, the number of myofibers efficientlyexpressing the transgene was determined (n=8 muscles per formulation andtemperature). Results are expressed on panel B on the histogram as mean±standarddeviation for both pluronic L64 (plain) and tetronic 304 (hatched) gene carriers. Theaverage number of levels for which transfected myofibers could be observed was alsodetermined for both pluronic L64 (▲) and tetronic 304 (●). Statistical significance wasassessed using Bonferroni t-test. ⁎pb0.05 = pluronic L64/DNA at 37 °C versus sameformulation at 4 °C and 20 °C.

Fig. 6. Transfection efficiency of pluronic L64 and tetronic 304 gene carriers on dystrophic muscles. Pluronic L64 and tetronic 304 formulations were prepared at 37 °C and directlyinjected into tibialis anterior (A) and quadriceps (B) muscles of CHF147 hamsters. Representative sections are presented for both formulations and the different types of skeletalmuscles transfected.

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4 °C to 37 °C. These results were confirmed by transmission electronmicroscopy experiments (data not shown).

The next step was to assess whether these modifications of theorganization of the polymer/DNA systems could influence theirtransfection efficiency in vivo. As the medium seemed to be animportant factor for the micellization of the pluronic L64, we firstcompared the expression of the reporter gene coding for the β-galactosidase after direct intramuscular injection of pluronic-basedformulations prepared in water, saline or Tyrode's salts solution(Fig. 4). When formulated in water (Fig. 4A) or saline (Fig. 4B), genecarriers based on pluronic L64 exhibited transfection efficiency lowercompared to the one prepared in Tyrode (Fig. 4C).

The effect of temperature was then assessed on pluronic L64 andtetronic 304 gene delivery systems (Fig. 5).

All formulations were prepared and maintained at constanttemperature until injection. One week after administration, transfec-tion efficiency of amphiphilic copolymers was determined byassessing the number of transfected myofibers at each of the tenlevels where cryo-sections were performed. Transgene expressionwas significantly more important for formulations based on pluronicL64 prepared at 37 °C compared to 4 °C and 20 °C. When consideringtetronic 304 based carriers, no statistical difference in the transgeneexpression could be recorded when varying the temperature. For allthe gene carriers and whatever the temperature, the average numberof transfected levels was relatively important, e.g. up to 9 levels on 10displayed transfected myofibers, representing a length of around7.2 mm.

The formulations exhibiting maximal transfection efficiency, e.g.amphiphilic copolymers prepared in Tyrode's salts solution at 37 °Cwere thereafter injected in both tibialis anterior and quadriceps ofdystrophic hamsters (Fig. 6).

In both proximal and distal skeletal muscles, direct intramuscularinjection of gene carriers based on either pluronic L64 or tetronic 304resulted in efficient expression of the reporter gene. Moreover,pluronic L64 and tetronic 304 were capable to promote transfectionin large as well as small or fragmented myofibers of the dystrophicmuscles (see highest magnification on Fig. 6).

4. Discussion

Amphiphilic block copolymers are focusing great interest in thefield of gene delivery, especially to transfer DNA to the striatedmuscles. However, the mechanism of action whereby they promote

tissue transfection remains yet to be understood. Due to theiramphiphilic properties, pluronic and tetronic copolymers display theability to form micelles under certain physical conditions.

In our studies, we could observe that the CMC of tetronic 304 isdecreasing when increasing the temperature, which is consistent withpreviously published data [12]. When increasing the temperature, thehydrophobicity of the PO blocks raises and its solubility in aqueoussolutions thus decreases, leading to the formation of micelles.Nevertheless, we could demonstrate that concentrations of tetronic304 utilized in a gene transfer purpose are far above the CMC,independently on the temperature considered. At temperaturessuitable for in vivo administrations, tetronic 304 is thus formingmicelles. The enthalpy of interaction between tetronic 304 andplasmid DNA was constant in the whole range of temperatures thatwe investigated. This effect can be related to the absence ofmodification in the structure of tetronic 304/DNA carriers. In aprevious study, we have demonstrated that when associated to DNA inTyrode, tetronic 304 leads to the formation of condensation nodessurrounded by unfolded loops of plasmids [20]. When comparing themicrographs obtained by transmission electron microscopy withtetronic 304/DNA systems prepared at various temperatures, nodifferences were recorded. In accordance with our biophysicalcharacterization, tetronic 304/DNA formulations did not exhibitsignificant differences in terms of in vivo transgene expression whenmodifying the temperature.

On the other hand, we showed that pluronic L64 in water formedmicelles when reaching temperatures above the physiological range.However, interactions could be recorded between the unimers at lowconcentration of pluronic L64 by changing the medium used toprepare the formulations. For pluronic L64, the medium used as wellas the temperature are strongly affecting the capacity of thisamphiphilic copolymer to form micelles at the concentrationsgenerally used to perform gene delivery. We thus showed thatunimers could interact at concentrations consistent with the in vivoadministration only when raising the temperature to 37 °C andpreparing the formulations in a Tyrode's salts solution. These data canbe related to experiments demonstrating that the CMC and the criticalmicelle temperature (CMT) of pluronic L64 are significantly alteredupon addition of salts [21,22]. When adding salt to the copolymersolution, a rearrangement of the water molecules occurs: watermolecules which are bound to the PO or the EO residues are releasedand thus interaction between the hydrophobic blocks of pluronicincreases, leading to a decrease of the CMC [23]. All these studies

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showed that the decrease of the CMC and CMT is more important inthe presence of Cl− compared to Br− or I− and for Na+ compared to K+

or Li+. However, in our study no micellization was recorded in salinesolution composed of 150mMNaCl at physiological temperature and inthe range of concentration of pluronic L64 investigated. Conversely,interactions between the unimers were observed at 37 °C when usingthe Tyrode's salts solution. Tyrode is a more complex solution thansaline. It is composedof NaCl and KCl (137 and 2.7mMrespectively), butit also contains divalent cations, e.g. Ca2+ and Mg2+ (CaCl2 1.8 mM andMgCl2 1.1 mM). This strongly suggests that these divalent cations couldhave a more pronounced effect on the decrease of the CMC withreference to Na+, K+ or Cl−.

We also demonstrated that this organization has a major influenceon the interactions that can occur between the copolymer and theDNA. Contrarily to our observations, a recent publication by Gau-Racine et al. [24] using small angle neutron scattering (SANS), showedthat repulsive interactions might exist between the DNA and thepluronic L64. However, their experiments were performed on smallfragments of linear, double stranded DNA and not on plasmid DNA.Plasmid DNA can exhibit various arrangements (supercoiled, coiled orrelaxed) that are not found with linear DNA and which might have animpact on the interactions developed with the polymer. Furthermore,in these experiments, preparationmedia differ significantly from ours.We would like to stress that the interaction between the polymer andthe DNA is largely influenced by the medium used for the preparation.Therefore, the type and topology of DNA as well as the preparationmedia might explain the discrepancy between both sets ofexperiments.

What is even more interesting is the impact of this particularorganization of the pluronic L64 on its transfection efficiency in vivo.We provide evidence that pluronic L64/DNA carriers exhibit highestefficiency to transfect skeletal muscles when prepared in Tyrode'ssalts solution at 37 °C. We thus showed that the supramolecularassembly of amphiphilic block copolymers is an important parameterto ensure maximal efficiency of the gene carrier. Moreover, tempera-ture andmedium are parameters that can be easily controlled in orderto optimize gene transfer.

Although pluronic L64 and tetronic 304 are both amphiphiliccopolymers composed of EO and PO blocks, their architecture isdifferent and tetronic displays one charge at physiological pH [25]. Theassociation between tetronic 304 and the DNA led to partialcondensation of the plasmids, as we previously showed [26], contra-rily to pluronic L64 which triggered no condensation of the DNA, evenwhen forming micelles. As we didn't noticed differences in the shapeof tetronic 304/DNA systems by transmission electron microscopy ordynamic light scattering when modifying the temperature, this mightalso explain that transfection efficiency of such carriers is notdepending on this parameter.

Finally, optimized formulations based on both tetronic 304 andpluronic L64 were demonstrated to induce efficient gene transfer tothe dystrophic muscles of CHF147 Syrian hamsters. CHF147 hamsterspresent a limb-girdle muscular dystrophy, a pathology triggeringmore severe dystrophic effects on proximal muscles like thequadriceps than on distal ones as tibialis anterior [27]. Despite thedisorganization of the muscle tissue and the presence of interstitialfibrosis which may represent an important hurdle to transfer DNA tothe myofibers, amphiphilic copolymers based systems achievedefficient transfection of both distal and proximal muscles. Moreover,these gene delivery systems seemed tomediate efficient expression ofthe transgene, independently of the shape of the myofibers (fromsmall, atrophic or fragmented to large ones). However, pluronic L64seems to induce less local lesions after direct intramuscular injections.This could be related to the sealant effect mediated by pluroniccopolymers on the membrane of damaged myofibers, a property thathas already been described for pluronic F68, also known as poloxamer188 [28–30].

5. Conclusion

Amphiphilic block copolymers belonging to the tetronic andpluronic families are focusing increasing interest in the field of genetherapy. Nevertheless, their mechanisms of action as well as theparameters that might influence their transfection efficiency remainlargely unknown. This works highlights the influence of the mediumand the temperature on the organization of pluronic L64 copolymer.We also show that interactions can occur between pluronic L64 andthe DNA, but only in the presence of micelles. The conditions leadingto interactions between unimers are also displaying maximalefficiency in vivo after direct intramuscular injection in both normaland dystrophic muscles.

Acknowledgements

The authors would like to acknowledge André Salmon for excellentanimal care and technical expertise on histological techniques. Thiswork was financially supported by the Association Française contre lesMyopathies (AFM).

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